大鼠骨髓间充质干细胞移植对Walker-256肝癌生长影响的实验研究

Effects of mesenchymal stem cell transplantation on growth of liver cancer ：experiment with rats

目的评价大鼠骨髓间充质干细胞（MSC）移植对Walker-256肝癌生长的影响。方法从大鼠腹水瘤株中提出并培养Walker-256细胞，采用直接肝内注射法制备大鼠肝癌模型。实验分2个实验组（MSC与肿瘤细胞混合移植组、MSC静脉移植组）和一个对照组，每组15只。所有实验动物均在术后第3、6、9、12天进行MR成像并测量肿瘤横断位最大层面面积的变化，第12天成像后取病理进行常规HE染色以及血管内皮细胞生长因子（VEGF）、nm23、增殖细胞核抗原（PCNA）、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）法免疫组化分析。结果MRI显示所有实验动物在术后第3、6天均未见明显肿瘤形成，第9天可见肿瘤结节生长，两个实验组肿瘤在第9、12天横断位最大层面面积均大于对照组（F=4．21，P〈0．05；F=8．52，P〈0．01）。免疫组化结果表明两个实验组肿瘤VEGF表达均明显高于对照组（F=9．58，P〈0．01），nm23基因蛋白表达均低于对照组（F=4．61，P〈0．05），MSC混合移植组PCNA表达高于对照组[d′（1,0.05）=0．34，d′（1,0.01）=0．63，P〈0．05]，MSC静脉移植组PCNA表达与对照组差异无统计学意义[d′（1,0.05）=0．32，d′（1,0.01）=0．48，P〉0．05]，2个实验组肿瘤凋亡指数与对照组差异均无统计学意义（F=1．25，P〉0．05）。结论大鼠MSC移植能影响Walker-256肝癌VEGF、nm23以及PCNA的表达，有利于肿瘤的生长。

Objective To evaluate the effects of mesenchymal stem cell （MSC） transplantation on the growth of liver cancer. Methods MSCs were isolated from the bone marrows of SD rats. Walker-256 cancer cells were isolated from the cancerous aseites of rat and cultured. Forty-five SD rats were randomly divided into 3 equal groups ： mixed transplantation group undergoing laparotomy and transplantation of cancer cells mixed with MSCs into the liver, MSC Ⅳ transplantation group undergoing injection of MSCs into the caudal vein, and control group undergoing only MSC transplantation into the liver. MR imaging was performed s at days 3, 6, 9 and 12 after modeling to measure the maximum cross section area of the tumor. At day 12 the rats were killed after MR imaging with their livers taken out to undergo HE staining and pathological examination. Immunohisochenmisry was used to detect the expression of vascular endothelial cell growth factors （VEGF）, nm23 gene, a tumor metastasis inhibiting gene, and proliferating cell nuclear antigen （PCNA） , a nuclear polypeptide necessary in the DNA synthesis. Results No significant evidence of tumor formation was detected by MRI at days 3 and 6 after modeling in all rats and tumor nodules were observed since day 9. The maximum cross section areas of tumor of the mixed transplantation group and MSC IV transplantation group were significantly larger than that of the control group at days 9 and 12 （ F =4. 21, P 〈 0. 05 ; F = 8.52, P 〈 0. 01 ）. hnmunohistochemistry showed that VEGF expression levels of the two study groups were both significantly higher than that of the control group （ F = 9. 58, P 〈 0. 01 ）, while the nm23 gene expression levels of the 2 study groups were both significantly lower than that of the control group （ F =4. 61, P 〈 0. 05 ）. The PCNA expression level of the mixed transplantation group was significantly higher than that of the control group （ d′（1,0.05） = 0. 34, d′（1,0.01） = 0. 63, P 〈 0. 05 ） , however, there was no significant difference in the PCNA expression level between the MSCs Ⅳ transplantation group and the control group （ d′〈1,0. 05） = 0. 32, d′（1,0.01） = 0. 48, P 〉 0. 05 ）. There was no significant difference in the tumor apoptotic index between the 2 study groups and the control group （ F = 1.25, P 〉 0. 05 ） . Conclusion MSC transplantation increases the expression of VEGF and PCNA, while decreases the expression of nm23 gene in cancer cells, thus favoring the tumor growth.